Neurotransmitters, receptors and their specific pathways and synapses make up the cytochemical substrate of the auditory system. Knowledge of this cytochemical substrate is needed to understand auditory function but is also crucial towards identification and treatment of dysfunctions. We have been working towards the goal of defining this substrate in the lower auditory brainstem for the last ten years, with studies of neurotransmitter candidates including excitatory and inhibitory amino acids and neuropeptides. Our approach has relied on immunocytochemical techniques at the light and electron microscopic levels and antibodies to neurotransmitter candidates, related enzymes, and associated receptors. Many of our studies represent the first examination in the auditory system (often among the first in any system) and recent findings include the first demonstration of GABA and glycine co-localization and of GABAa/BZD receptor heterogeneity. We have also carried out several types of tract tracing studies (anterograde, retrogade and lesion). The proposed studies are to continue towards the goal of defining the cytochemical substrate of the auditory brainstem, addressing the major issues of co-localization of neurotransmitters and receptor heterogeneity and combining tract-tracing and immunocytochemistry. We propose to: 1) investigate the distribution of GABA and glycine immunoreactive terminals in the lower auditory brainstem; determine their relative numbers, which terminals co-contain GABA and glycine and elucidate how GABA, glycine and GABA and glycine co-labeled immunoreactive terminals relate to immunolabeled glycine and GABA receptors, 2)determine the distribution of excitatory amino acid cells and terminals in the lower auditory brainstem using antibodies to glutamate and glutaminase, 3)determine how GABA, glycine and glutamate immunoreactive terminals are distributed on somata and dendritic trees of cochlear nucleus spherical bushy cells and octopus cells, 4)determine the sources and subclasses of GABA, Gly and Glu immunoreactive terminals on defined cells types in ventral cochlear nucleus (CN), lateral superior olive (LSO) and medial nucleus of the trapezoid body (MNTB) through combined immunocytochemical-tract tracing, 5)investigate heterogeneity in the expression of the GABA receptor in defined cell types in the CN using cDNA probes and monoclonal antibodies to the different subunits of the GABA receptor in in situ hybridization and immunocytochemical studies.